ABSTRACT
Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.
Subject(s)
Immunotherapy, Adoptive , T-Lymphocytes , Immunotherapy, Adoptive/methods , Immunotherapy , Receptors, Antigen, T-CellABSTRACT
CAR T cell therapy is a rapidly growing area of oncological treatments having a potential of becoming standard care for multiple indications. Coincidently, CRISPR/Cas gene-editing technology is entering next-generation CAR T cell product manufacturing with the promise of more precise and more controllable cell modification methodology. The intersection of these medical and molecular advancements creates an opportunity for completely new ways of designing engineered cells to help overcome current limitations of cell therapy. In this manuscript we present proof-of-concept data for an engineered feedback loop. We manufactured activation-inducible CAR T cells with the help of CRISPR-mediated targeted integration. This new type of engineered T cells expresses the CAR gene dependent on their activation status. This artifice opens new possibilities to regulate CAR T cell function both in vitro and in vivo. We believe that such a physiological control system can be a powerful addition to the currently available toolbox of next-generation CAR constructs.
Subject(s)
Receptors, Chimeric Antigen , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , CRISPR-Cas Systems , Gene Editing/methods , Immunotherapy, Adoptive/methods , T-Lymphocytes/metabolismABSTRACT
T cell activation is a cornerstone in manufacturing of T cell-based therapies, and precise control over T cell activation is important in the development of the next generation T-cell based therapeutics. This need cannot be fulfilled by currently available methods for T cell stimulation, in particular not in a time dependent manner. Here, we describe a modular activation reagent called Expamers, which addresses these limitations. Expamers are versatile stimuli that are intended for research and clinical use. They are readily soluble and can be rapidly bound and removed from the cell surface, allowing nearly instantaneous initiation and termination of activation signal, respectively. Hence, Expamers enable precise regulation of T cell stimulation duration and provide promise of control over T cell profiles in future products. Expamers can be easily adopted to different T cell production formats and have the potential to increase efficacy of T cell immunotherapeutics.
Subject(s)
Indicators and Reagents/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , Animals , Cell Proliferation , Gene Expression Profiling , Humans , Immunotherapy, Adoptive , Mice , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high)/CD25(high)/CD45RA(high) 'regulatory T cells' and CD8(high)/CD62L(high)/CD45RA(neg) 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.
Subject(s)
Antigens, CD/analysis , Cell Separation/methods , Flow Cytometry/methods , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Adult , Antigens, CD/immunology , CD4 Antigens/analysis , CD4 Antigens/immunology , CD8 Antigens/analysis , CD8 Antigens/immunology , Female , Humans , Immunoglobulin Fab Fragments/analysis , Immunoglobulin Fab Fragments/immunology , Interleukin-2 Receptor alpha Subunit/analysis , Interleukin-2 Receptor alpha Subunit/immunology , L-Selectin/analysis , L-Selectin/immunology , Leukocyte Common Antigens/analysis , Leukocyte Common Antigens/immunology , Male , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunologyABSTRACT
A retrospective comparison of WT1 and BCR-ABL1 expression was performed in 40 imatinib-treated chronic myeloid leukaemia patients. The overall correlation of WT1 and BCR-ABL1 was low. In two patients WT1 expression was increasing despite very low BCR-ABL1 levels. As both revealed Ph-negative aberrant clones, a second independent cohort of 20 cases, all with Ph-negative clonal evolution, was analysed. High WT1 expression (5.0-177.0%) was detected in a case with +11 and in four of eight cases with +8, but not in cases with del(20q) or -Y. Thus, increasing WT1 levels in molecular responders may indicate Ph-negative clonal cytogenetic evolution during imatinib treatment.
Subject(s)
Antineoplastic Agents/therapeutic use , Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/therapeutic use , Pyrimidines/therapeutic use , WT1 Proteins/metabolism , Adult , Aged , Benzamides , Cohort Studies , Female , Fusion Proteins, bcr-abl/genetics , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Male , Middle Aged , Polymerase Chain Reaction , Retrospective Studies , WT1 Proteins/genetics , Young AdultABSTRACT
Nucleophosmin (NPM1)-mutated acute myeloid leukemia (AML), which is recognized as a provisional entity in the World Health Organization 2008 classification of myeloid neoplasms, accounts for 30% of AML. We analyzed 1227 diagnostic and follow-up samples in 252 NPM1-mutated AML patients with 17 different NPM1 mutation-specific real-time quantitative polymerase chain reaction (RQ-PCR) assays. Paired diagnostic/relapse samples of 84 patients revealed stable NPM1 mutations in all cases, suggesting that they are pathogenetically early events and thus applicable for minimal residual disease detection. A total of 47 relapses were predictable because of an NPM1 mutation level (%NPM1/ABL1) increase of at least 1 log or in 15 cases because of NPM1 mutation levels not decreasing less than 3 log ranges. A high prognostic value of NPM1 levels was shown for 4 different intervals after therapy was initiated. Furthermore, thresholds of 0.1 and 0.01%NPM1/ABL1 during/after treatment discriminated between prognostic subgroups. Univariate analyses, including age, white blood cell count, blast count, CD34 positivity, FLT3 mutations status, FAB type, karyotype, NPM1 mutation type, and pretreatment NPM1 mutational level, showed that, besides NPM1 mutation level, only age and FLT3-LM mutation status were prognostically significant for EFS. Multivariate analysis, including age, FLT3-LM status, and NPM1 mutation level at different time points, demonstrated that NPM1 level was the most relevant prognostic factor during first-line treatment. Similar results were obtained in patients undergoing second-line chemotherapy or allogeneic stem cell transplantation.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Mutation , Nuclear Proteins/genetics , Polymerase Chain Reaction , Adult , Age Factors , Aged , Antigens, CD34/genetics , Antigens, CD34/metabolism , Female , Follow-Up Studies , Humans , Leukemia, Myeloid, Acute/blood , Leukemia, Myeloid, Acute/therapy , Leukocyte Count , Male , Middle Aged , Neoplasm, Residual , Nuclear Proteins/metabolism , Nucleophosmin , Prognosis , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Recurrence , Stem Cell Transplantation , Transplantation, Homologous , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolismSubject(s)
Chromosomes, Human, Pair 3 , DNA-Binding Proteins/genetics , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Proto-Oncogenes/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Transcription Factors/genetics , Bone Marrow/metabolism , Bone Marrow Cells/metabolism , Chromosome Aberrations , Humans , Karyotyping , Leukocytes, Mononuclear/metabolism , MDS1 and EVI1 Complex Locus Protein , Myelodysplastic Syndromes/genetics , Oncogene Proteins, Fusion/genetics , Sensitivity and Specificity , Transcription, GeneticABSTRACT
Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3-length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1-/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM- mutations are an independent predictor for a favorable outcome in AML with normal karyotype.